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Abstract:

A conjugate is provided for cell processing, which comprises a magnetic
particle and a surface modifier having specific affinity to a target
cell. The particle and modifier are linked through a cleavable peptide
bond. In a method of cell processing, the conjugate is attached to a
target cell; the target cell attached to the conjugate is subject to
magnetic processing; the peptide bond is cleaved to separate the
processed target cell from the magnetic particle; the target cell
separated from the magnetic particle is attached to a substrate. The
magnetic particle may include an iron oxide, and the surface modifier may
include a glucosamine. The particle and modifier may be linked by a
linker comprising a protease recognition site and a peptide bond. The
linker links the surface modifier to the particle, and cleavage of the
peptide bond is catalyzed by a specific protease that recognizes the
protease recognition site.

Claims:

1. A conjugate comprising: a magnetic particle comprising an iron oxide;
a surface modifier comprising a glucosamine; and a linker comprising a
protease recognition site and a peptide bond, wherein said linker links
said surface modifier to said particle, and wherein cleavage of said
peptide bond is catalyzed by a specific protease that recognizes said
protease recognition site.

2. The conjugate of claim 1, wherein said protease is thrombin.

3. The conjugate of claim 1, wherein said particle comprises a quantum
dot.

4. The conjugate of claim 1, wherein said particle is a nanoparticle.

5. The conjugate of claim 1, wherein said particle is superparamagnetic.

9. A method of cell processing, comprising: attaching a conjugate to a
target cell, said conjugate comprising a magnetic particle, a surface
modifier having a specific affinity to said target cell, wherein said
particle and modifier are linked through a cleavable peptide bond;
subjecting said target cell attached to said conjugate to magnetic
processing; cleaving said peptide bond to separate said target cell from
said magnetic particle; and providing a substrate and allowing said
target cell separated from said magnetic particle to attach to said
substrate.

10. The method of claim 9, wherein said conjugate comprises a linker
linking said surface modifier to said magnetic particle, said linker
comprising a protease recognition site and said peptide bond, wherein
cleavage of said peptide bond is catalyzed by a specific protease that
recognizes said protease recognition site, and wherein said cleaving
comprises exposing said linker to said protease.

11. The method of claim 10, wherein said protease is thrombin.

12. The method of claim 9, wherein said surface modifier comprises a
glucosamine, glutamine, or galactose.

13. The method of claim 9, wherein said magnetic particle comprises a
quantum dot or a nanoparticle.

14. The method of claim 9, wherein said magnetic particle is
superparamagnetic.

15. A method of cell processing, comprising: attaching the conjugate of
claim 1 to a target cell; subjecting said target cell attached to said
conjugate to magnetic processing; cleaving the peptide bond in said
conjugate to separate said target cell from the magnetic particle in said
conjugate; and providing a substrate and allowing said target cell
separated from said magnetic particle to attach to said substrate.

17. A method of forming a conjugate for attachment to a cell, comprising:
linking a surface modifier to a magnetic particle with a linker to form
the conjugate; wherein said surface modifier is selected to have a
specific affinity to said cell; and wherein said linker is selected such
that said linker comprises a protease recognition site and a peptide
bond, and cleavage of said peptide bond is catalyzed by a specific
protease that recognizes said protease recognition site.

18. The method of claim 17, wherein said protease is thrombin.

19. The method of claim 17, wherein said surface modifier comprises a
glucosamine, glutamine, or galactose.

20. The method of claim 17, wherein said magnetic particle comprises a
quantum dot or a nanoparticle.

21-25. (canceled)

Description:

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of and priority from Singapore
Patent Application No. 2010002272-1, filed Mar. 31, 2010 and entitled
"Glucosamine-conjugated Iron Oxide Nanoparticles for the Separation of
Insulin Secreting Beta Cells," the entire contents of which are
incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to conjugates for cell manipulation
and processing, use of the conjugates, and methods of cell manipulation
and processing.

BACKGROUND OF THE INVENTION

[0003] The movement of cells may be controlled by binding magnetic
particles to target cells and applying a magnetic field to move the
magnetic particles and thus the target cells bonded to the magnetic
particles. Such techniques may be used in cell processing, such as cell
manipulation, cell separation, cell sorting, or other applications where
control of cell movement is needed. Such techniques are thus useful in a
wide variety of biomedical applications, tissue engineering, and other
processes involving the use of cells. For example, cell separation may be
used to remove unwanted cells, to collect desired cells, to purify a cell
population, or to control the cell environment. Magnetic particles bonded
to cells may also be used to mark or label cells for cell detection or
magnetic imaging.

[0004] Cellular adhesion is the binding of a cell to a surface,
extracellular matrix or another cell, typically mediated by cell adhesion
molecules such as cell surface proteins that are selectins, integrins, or
cadherins. Cellular adhesion is an aspect of cellular growth and
multiplication for many cell types (Gumbiner, B. M., "Cell adhesion: The
molecular basis of tissue architecture and morphogenesis," Cell, (1996),
vol. 84, pp. 345-357).

SUMMARY OF THE INVENTION

[0005] In one aspect, the invention provides a conjugate that may be used
to facilitate the magnetic processing of cells, the conjugate having a
linker that may be cleaved to facilitate subsequent cellular processes,
such as cellular adhesion. In selected embodiments, a conjugate disclosed
herein comprises a magnetic particle and a surface modifier having a
specific affinity to target cells. The particle and the modifier are
linked through a cleavable peptide bond specific to a protease.

[0006] The conjugates can attach to the target cells and can be used for
cell processing, such as cell sorting or cell separation with magnetic
force, and magnetic imaging or detection. The magnetic particles can be
conveniently separated from the target cells after initial processing and
before attaching the cells to a substrate, by exposing the processed
target cells to the specific protease to cleave the peptide bonds, thus
severing the links between the magnetic particles and the cells.
Subsequently, the target cells separated from the magnetic particles can
be conveniently attached to the substrate, without interference from the
magnetic particles.

[0007] Thus, in accordance with an aspect of the present invention, there
is provided a conjugate comprising a magnetic particle comprising an iron
oxide; a surface modifier comprising a glucosamine; and a linker
comprising a protease recognition site and a peptide bond. The linker
links the surface modifier to the particle, and cleavage of the peptide
bond is catalyzed by a specific protease that recognizes the protease
recognition site. The protease may be thrombin. The magnetic particle may
comprise a quantum dot. The particle may be a nanoparticle. The particle
may be superparamagnetic. The particle may comprise magnetite. The linker
may comprise a protease recognition sequence. The protease recognition
sequence may comprise Leu-Val-Pro-Arg-Gly-Ser.

[0008] In accordance with a further aspect of the present invention, there
is provided a method of forming a conjugate as described in the preceding
paragraph, comprising linking the surface modifier to the magnetic
particle with the linker.

[0009] In accordance with another aspect of the present invention, there
is provided a method of cell processing. In this method, a conjugate is
attached to a target cell, where the conjugate comprises a magnetic
particle and a surface modifier having a specific affinity to the target
cell. The particle and modifier are linked through a cleavable peptide
bond. The target cell attached to the conjugate is then subject to
magnetic processing. The peptide bond is cleaved to separate the target
cell from the magnetic particle. A substrate is provided and the target
cell separated from the magnetic particle is allowed to attach to the
substrate. The conjugate may comprise a linker linking the surface
modifier to the magnetic particle, wherein the linker comprises a
protease recognition site and the peptide bond, and cleavage of the
peptide bond is catalyzed by a specific protease that recognizes the
protease recognition site. Cleaving the peptide bond may comprise
exposing the linker to the protease. The protease may be thrombin. The
surface modifier may comprise a glucosamine, glutamine, or galactose. The
magnetic particle may comprise .a quantum dot or a nanoparticle. The
magnetic particle may be superparamagnetic. The magnetic processing may
comprise magnetically sorting or separating cells. The conjugate may be
any conjugate disclosed herein.

[0010] In accordance with a further aspect of the present invention, there
is provided a method of forming a conjugate for attachment to a cell. The
method comprises linking a surface modifier to a magnetic particle
through a linker to form the conjugate. The surface modifier is selected
to have a specific affinity to the cell. The linker is selected such that
it comprises a protease recognition site and a peptide bond, and cleavage
of the peptide bond is catalyzed by a specific protease that recognizes
the protease recognition site. The protease may be thrombin. The surface
modifier may comprise a glucosamine, glutamine, or galactose. The
magnetic particle may comprise a quantum dot or a nanoparticle. The
conjugate may be any conjugate disclosed herein.

[0011] In accordance with another aspect of the present invention, a
conjugate disclosed herein is used in the processing of cells, such as
magnetically sorting or separating the cells.

[0012] Other aspects and features of the present invention will become
apparent to those of ordinary skill in the art upon review of the
following description of specific embodiments of the invention in
conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In the figures, which illustrate, by way of example only,
embodiments of the present invention,

[0014] FIG. 1 is a schematic diagram of a conjugate, exemplary of an
embodiment of the present application;

[0015]FIG. 2 is a schematic diagram of a chemical reaction for forming
maleimidoglucosamine;

[0016]FIG. 3 is a schematic diagram of a chemical reaction for forming a
glucosamine-peptide complex;

[0017]FIG. 4 is a flow chart for a process of cell separation, exemplary
of an embodiment of the present application;

[0018]FIG. 5 is a flow chart for cell processing, exemplary of an
embodiment of the present application;

[0019] FIG. 6 is a schematic diagram for the synthesis route of forming
comparison conjugates;

[0020] FIG. 7 is a transmission electron microscopy (TEM) image of the
comparison conjugates formed according to the synthesis route of FIG. 6;

[0021]FIG. 8 is a TEM image of sample iron oxide nanoparticles used for
forming the conjugates of FIG. 7;

[0032]FIG. 22 is a bar graph showing the percentage of cells in samples
incubated with conjugates having peptide linker and conjugates having no
peptide linker respectively; and

[0033] FIGS. 23 and 24 are images of culture substrates after cell culture
with the respective sample cells of FIG. 22.

DETAILED DESCRIPTION

[0034] An exemplary embodiment of the present invention is a conjugate 100
of a magnetic particle 102 and a surface modifier 104, as illustrated in
FIG. 1. Particle 102 and modifier 104 are linked by a severable linker
106.

[0035] Magnetic particle 102 may be a nanoparticle. Nanoparticles
typically refer to particles having a particle size of about 1 to about
100 nm. In some embodiments, particle 102 may have a particle size of
about 6 to about 8 nm. In alternative embodiments, the particle size may
be from about 2 to about 20 nm. In further embodiments, the particle size
may be about 50 nm. The particle size may also be larger, such as from
about 100 nm to a few micrometers. In one embodiment, the particle size
may be about 150 nm, or larger than 2 μm. Other particle sizes may
also be selected depending on the particular application. Particle 102
may have any shape, such as a generally spherical, generally cubic, or
irregular shape. In some applications, the shapes and sizes of the
particles used may be substantially uniform, and may be controlled for a
particular purpose. In other applications, the sizes or shapes of the
particles may vary. The term "particle size" as used herein refers to the
average diameter of the particle when the particle has a generally
spherical shape. As particles may have non-spherical shapes and different
sizes, the particle size refers to the average size of the particles when
used in reference to multiple particles. When a particle has an irregular
non-spherical shape, its particle size refers to its effective diameter,
which is the diameter of a spherical particle that has the same volume as
the non-spherical particle. In cases where the particle has a generally
geometrical shape, such as a cubic shape, the particle size may refer to
a characteristic dimension for that geometrical shape. For example, a
cubic shape may be characterized by the length of its side.

[0036] Particle sizes and size distribution of particles can be measured
using optical or electronic imaging techniques, such as transmission
electron microscopy (TEM) or suitable light scattering (e.g. dynamic
light scattering) techniques. Such techniques can be readily understood
and applied by persons skilled in the art for a given application. The
average particle size may be determined using standard techniques, for
example, by measuring the size of a representative number of particles.

[0037] Particle 102 is formed of a magnetic material such that its
movement can be controlled by applying a magnetic force, the benefits of
which will become apparent below. The magnetic material may be
ferromagnetic, or superparamagnetic. In some embodiments, particle 102
may be formed of an iron oxide, such as magnetite (Fe3O4). As
can be appreciated, magnetite is more magnetic and magnetite particles
may be conveniently manipulated with a weaker magnetic force, as compared
to particles formed of other forms of iron oxides. However, in some
embodiments, other forms of magnetic iron oxides may also be used.
Possible other forms of iron oxides may include FeO,
α-Fe2O3, β-Fe2O3, γ-Fe2O3,
and ε-Fe2O3. For example, a superparamagnetic iron
oxide may be used. In one embodiment, maghemite (γ-Fe2O3)
may be used. A mixture of different iron oxides may also be used. For
example, a mixture of magnetite and maghemite may be included in particle
102.

[0038] In some embodiments superparamagnetic iron oxide (SPIO)
nanoparticles may be used. For example, ultrasmall superparamagnetic iron
oxide nanoparticles (USPIO), which have an average individual particle
size of about 10 to 40 nm, may be used in some embodiments. The USPIO may
be monocrystalline iron oxide nanoparticles (MION) with an average
particle size of about 10 to about 30 nm. The SPIO nanoparticles may also
have particle sizes from about 60 to about 150 nm, or from about 300 nm
to about 3.5 μm, depending on the particular application. Particle 102
may include a single iron oxide crystal, or multiple iron oxide crystals.
As can be appreciated by those skilled in the art, single-crystal
particles have some properties that are not present in multi-crystal
particles, which may conveniently provide certain benefits in some
applications.

[0039] It is not necessary that particle 102 is entirely formed of a
magnetic material. Particle 102 may include other materials that are
specifically included for a desired function or materials that are
incidentally included during manufacturing or processing. For example, a
surface treatment material may be applied to the particle surface to
modify, e.g., the solubility of the particle in a given solvent such as
water. For instance, particle 102 may include a hydrophilic polymer
coating. Particle 102 may also include a component material for labeling
or imaging purposes. For instance, an optical label or marker such as a
fluorescent material may be included in particle 102. In some
embodiments, particle 102 may be an aggregate of two or more smaller
individual particles. The different individual particles may be formed of
the same material or different materials. For instance, particle 102 may
be a heterodimer particle.

[0040] Surface modifier 104 is formed of one or more small molecules that
have specific binding affinities to selected target cells, and is used to
modify the particle surface so that the modified particle can selectively
attach to selected target cells, the benefits of which will become
apparent below. A small molecule is not a polymer and has a relatively
low molecular weight. Typically, small molecules have a molecular weight
of less than 800 Da. Small molecules can bind with high affinity to a
biopolymer such as protein, nucleic acid, or polysaccharide, and, when
attached the biopolymer, may alter the activity or function of the
biopolymer. Two or more surface modifying molecules may be linked to each
particle 102, as illustrated in FIG. 1. The target cells may be insulin
secreting beta cells, hepatocyte cells, neuron cells, or other cells
having specific affinity to a small molecule. The surface modifier may be
selected so that it has an affinity to a cell surface marker that is not
internalized by the cell.

[0041] In the exemplary embodiment, surface modifier 104 includes a
glucosamine. The surface modifier 104 may be formed from maleimido
glucosamine, 2-Amino-2-deoxy-D-glucose hydrochloride, Chitosamine
hydrochloride, D-(+)-Glucosamine hydrochloride, N-Acetyl-D-glucosamine,
D-Glucosamine 6-sulfate, D-Glucosamine 6-phosphate, or the like.
Derivatives or variations of the above listed chemicals may also be used
as long as the amine functional group is retained.

[0042] A glucosamine can be an efficient surface modifier for specific
attachment to certain cells such as insulin-secreting beta cells and for
separating such cells from other cells. Without being limited to any
particular theory, it is expected that a glucosamine can bind to the
glucose transporter Glut2. As Glut2 is specifically expressed in certain
cells such as in insulin-secreting beta cells but not in other cells, a
glucosamine has specific binding affinity to insulin-secreting beta cells
or cells in which Glut2 is expressed. It has been reported in the
literature that Glut2 has a higher affinity for glucosamine than for
glucose.

[0043] As can be appreciated, other similar molecules such as glutamine or
galactose also have specific affinity to certain types of cells and may
also be used as surface modifiers. However, for attachment to cells which
express Glut2 receptors such as beta cells, a glucosamine surface
modifier can provide a high attachment efficiency and selectivity, as it
has high affinity to Glut2 but low affinity to other cells that do not
express Glut2 receptors. In contrast, galactose and glutamine do not have
high affinity to beta cells, as their corresponding receptors are not
generally expressed in beta cells.

[0044] Linker 106 has a protease recognition site and includes a peptide
bond, such that cleavage of the peptide bond is catalyzed by a specific
protease that recognizes the protease recognition site. In other words,
linker 106 includes a cleavable peptide bond specific to a selected
protease. The cleavage (breaking up) of a peptide bond specific to a
protease will be catalyzed by the specific protease. Linker 106 links
particle 102 and modifier 104 through the cleavable peptide bond, and is
selected such that when conjugate 100 is exposed to the specific
protease, cleavage of the peptide bond is catalyzed to sever the link
between particle 102 and modifier 104. The benefits of providing a
protease-specific peptide bond in the link will become apparent below.

[0045] Suitable molecules for linker 106 include, for example, small
molecules having a specific recognition sequence recognized by a selected
protease. For example, a primary recognition sequence for thrombin may be
expressed as P4-P3-Pro-Arg/Lys-cut-P1'-P2'[SEQ ID NO:
1] where P3 and P4 are hydrophobic and P1' and P2'
are non-acidic. Examples of such recognition sequences include
Leu-Val-Pro-Arg-cut-Gly-Ser [SEQ ID NO: 2] (pGEX-T vectors),
Met-Tyr-Pro-Arg-cut-Gly-Asn [SEQ ID NO: 3], and
Ile-Arg-Pro-Lys-cut-Leu-Lys [SEQ ID NO: 4] (inexact). A secondary
recognition sequence for thrombin may be expressed as
P2-Arg/Lys-cut-P1', where either P2 or P1' is Gly.
For example, a secondary recognition sequence may be Ala-Arg-cut-Gly or
Gly-Lys-cut-Ala. In the above expressions, the possible cleavage sites
are indicated by `cut`; and when a residue can be one of two amino acids
a slash (/) is used to separate the two possibilities. In one embodiment,
thrombin is the selected protease, and linker 106 comprises a recognition
sequence for thrombin, such as a sequence described above. For instance,
linker 106 may include the sequence of
cys-Leu-Val-Pro-Arg-Gly-Ser-gly-cys-gly [SEQ ID NO: 5].

[0046] For serine proteases (includes trypsin), linker 106 may include a
recognition sequence of LIVMSTASTAGHC [SEQ ID NO: 6], in which case, the
protease cuts at H. For cysteine proteases such as Tobacco Etch Virus
(TEV), linker 106 may include a recognition sequence of ENLYFQ(G/S) [SEQ
ID NO: 7], in which case, cleavage occurs between the Gln and Gly/Ser
residues. In selected embodiments, a linker 106 may be selected so that
it is susceptible to a protease that does not adversely impact a function
of a cell to which the conjugate is attached. For example, linker 106 my
be selected so that it is susceptible to a protease that does not cleave
cell surface domains of particular proteins, such as proteins that are
required for cellular adhesion or signaling.

[0048] As can be understood by those skilled in the art, in some
embodiments the protease recognition site may be the site at which
cleavage of the linker takes place. However, in other embodiments the
protease recognition site may be different from the site at which
cleavage of the linker occurs.

[0049] Linker 106 should be suitable for attachment to particle 102,
either chemically or physically. Linker 106 may include a terminal group
that can bind with the surface of particle 102.

[0050] Modifier 104 and linker 106 may be chemically bonded, and may be
provided in a single molecule. The modifier and the linker may also be
attached to one another through physical bonding.

[0051] A further exemplary embodiment of the present invention relates to
a process for preparing a conjugate such as conjugate 100. While
conjugate 100 may be formed according to the processes described herein,
it may also be prepared by other processes as will be understood by those
skilled in view of present disclosure.

[0052] In an exemplary process, particle 102 may be prepared using any
suitable technique. For example, suitable techniques for making magnetic
particles comprising magnetite are known to those skilled in the art.
Exemplary suitable techniques are disclosed in N. R. Jana et al., Chem.
Mater., 2004, vol. 16, p. 3931-3935 (referred to herein as "Jana"); J.
Park et al., Nat. Mater., 2004, vol. 3, p. 891-895 (referred to herein as
"Park"); or M. V. Kovalenko et al., J. Am. Chem. Soc, 2007, vol. 129, p.
6352-6353 (referred to herein as "Kovalenko"), the entire contents of
each of which are incorporated herein by reference. A specific example is
also described in Example I below. Magnetite nanoparticles with different
sizes and shapes may be prepared by changing experimental conditions,
such as reaction temperature, and the surfactant type used in the
process, and concentrations of different reagents. For instance,
spherical particles may be prepared by using oleic acid as the surfactant
and cubic particles may be prepared by using sodium oleate as the
surfactant. The preparation conditions may be adjusted according to the
procedures described in Jana, Park and Kovalenko.

[0053] Suitable magnetic particles may also be obtained from various
commercial sources. For example, suitable magnetic particles may be
obtained from Miltenyi Biotec®, Stemcell Technologies®,
Invitrogen®, or the like. The raw materials obtained from a commercial
source may be used directly or may be further treated before use.

[0054] Surface modifier 104 such as a suitable glucosamine may also be
prepared by any process known to skilled person in the art for forming
glucosamine. Surface modifier 104 or its precursor material may be
obtained from commercial sources such as from Sigma Aldrich®,
Merck®, or the like. A specific exemplary synthesis route for
preparing a suitable modifier is shown in FIG. 2, and described in
Example IIA.

[0055] Suitable severable linker materials or their precursor materials
may be obtained from commercial sources, such as Genescript®. Linker
materials may also be prepared according to known techniques for
preparing peptide materials.

[0056] The precursors for modifier 104 and linker 106 may be initially
reacted to form a modifier-linker complex. A specific example is shown in
FIG. 3, and described in Example IIB. The linker in the modifier-linker
complex is then bonded to the surface of particle 102. The procedures for
forming the complex and bonding it to the particle will depend on the
particular materials used and can be determined by those skilled in the
art. Specific exemplary procedures are described in Examples II and III
below.

[0057] The conjugates described herein can be used to process and
manipulate cells. In an exemplary embodiment, conjugate 100 may be used
for separating target cells from non-target cells, as illustrated in the
process S200 of FIG. 4. As will become apparent, in process S200 and
similar procedures involving manipulation of cells, conjugate 100 may be
replaced with other conjugates of magnetic particle and surface modifier
having specific affinity to the target cell, where the particle and the
modifier are linked by a linker that contains a cleavable peptide bond
specific to a protease. However, for simplicity of description, conjugate
100 is used below to represent all such conjugates unless otherwise
specified. It is also noted that multiple conjugates each having the
general structure of conjugate 100 are collectively referred to herein as
conjugates 100.

[0058] At S202, a mixture of target cells and non-target cells is
obtained. Such mixtures are common from normal cell sources in practice.
However, it is often desirable to separate the target cells from the
non-target cells for various reasons as understood by those skilled in
the art. As can be understood, sometimes it is not known if a cell sample
obtained from a given source contains a mixture of cell types. Such
samples may also be treated according to process S200 to remove
potentially present non-target cells. The cell mixture may be provided in
a solution such as an aqueous solution so that the cells are free to move
about.

[0059] At S204, conjugates 100 are dispersed in the cell mixture to allow
the conjugates to selectively attach to target cells due to the specific
affinity of the surface modifier 104 to the target cells.

[0060] Attachment of conjugates 100 to the target cells may be effected by
bonding between modifier 104 and a receptor on the cell surface. For
example, if Glut2 is expressed in the target cells, and the surface
modifiers of the conjugates contain glucosamine, glucosamine can bind
with Glut2 in the target cells.

[0061] Conjugates 100 are less likely to attach to non-target cells as
they have less affinity to bind with the non-target cells, as compared to
target cells. As can be appreciated, it is not necessary that all target
cells are bonded to conjugates 100 and all non-target cells are not
bonded to conjugates 100. As long as more target cells than non-target
cells are bonded with conjugates 100, the percentage of target cells in
total cells in the cell population can be increased using the process
S200 and some benefits can be obtained. Of course, as can be appreciated
by those skilled in the art, when the difference in binding affinity of
modifier 104 to target cells and non-target cells is larger, the
separation efficiency can be increased.

[0062] At S206, as conjugates 100 attached to the target cells are
magnetic, the target cells may be conveniently manipulated using a
magnetic force. For example, a magnetic field may be applied to the cell
mixture. The non-target cells that are not bonded with conjugates 100 or
another magnetic material will not be subject to the same magnetic force,
and as a result, their movement will be different from the movement of
the target cells bonded with conjugates 100 under the magnetic field.
This effect can be utilized to separate or sort the target cells.

[0063] For example, when the cells are suspended in a solution, a magnetic
force may be applied to force the target cells to move in a given
direction while the non-target cells stay in place.

[0064] In another example, a magnetic force may be applied to hold the
target cells in place and a fluid flow may be used to flush out the
non-target cells.

[0065] In some embodiments, cell separation may be effected with the use
of a magnetic column as illustrated in the Examples, and as can be
understood by those skilled in the art. For instance, the cells may be
separated using the magnetic-activated cell sorting (MACS) technique
known to persons skilled in the art. Cell separation and purification may
also be effected using a flow cytometry technique, which is also known to
persons skilled in the art.

[0066] Other techniques for cell separation with a magnetic force may also
be used as understood by those skilled in the art.

[0067] At S208, the separated target cells are collected. The collected
cell population will have a higher purity of target cells as compared to
the original cell mixture.

[0068] Either before or after S208, target cells may also be conveniently
subject to other types of magnetic processing. Magnetic processing may
include any process that utilizes the magnetic properties of the magnetic
particles attached to the target cells. Exemplary magnetic processing
includes magnetic detection, magnetic imaging, manipulation with magnetic
force, or the like. For example, superparamagnetic iron oxide
nanoparticles are expected to be good T2 contrast-enhancing agents, if
the conjugates contain magnetite nanoparticles, the target cells may be
conveniently studied or analyzed using a magnetic resonance imaging (MRI)
technique.

[0069] At S210, the target cells are exposed to water and the specific
protease that will catalyze cleavage of the peptide bond in the linker
106. For example, for linkers containing glucosamine, the protease may be
thrombin as the peptide bonds in glucosamine are specific to thrombin.

[0070] As can be understood by those skilled in art, peptide bonds can be
cleaved, or broken, by amide hydrolysis in the presence of water. Amide
hydrolysis of peptide bonds may occur spontaneously but the reaction is
very slow in normal conditions and in the absence of an enzyme that
catalyzes the hydrolysis reaction.

[0071] When the cleavage of the peptide bond is catalyzed by the protease,
severance of the link between the magnetic particle and the target cell
can occur within a practical period of time, such as from about to 15 to
60 minutes, or within about 30 minutes.

[0072] As can be appreciated, when peptide bonds specific to a protease
are used in linker 106, severance of the linker can be conveniently
controlled. When the specific protease is not present, cleavage of linker
106 is unlikely to occur quickly under normal conditions even if water is
present. Thus, the magnetic particles can remain attached to the target
cells for extended periods of time and during magnetic processing if
conjugates 100 are not exposed to the specific protease. The specific
protease can be mixed with the target cells attached to conjugates 100 in
an aqueous environment such as an aqueous solution, when it is the
desired time to sever the link between the magnetic particles and the
target cells.

[0073] Severance of the link can be confirmed, for example, by applying a
magnetic field to the cell population and observing the movement of the
target cells. If the movement of the target cells is unaffected by the
applied field, it indicates that the link with the magnetic particles has
been severed.

[0074] The target cells released from the magnetic particles can be
collected under a magnetic field, as the released cells will move
differently from those cells that are still attached to magnetic
particles in the magnetic field.

[0075] At S212, the released target cells are attached to a culture
substrate. This attachment may be effected using any suitable techniques
known to those skilled in the art. As the target cells are no longer
bonded to magnetic particles 102, interference from such particles can be
conveniently avoided. A culture substrate can be any supporting structure
on which cells can be cultured. For example a culture substrate may be a
culture plate, a culture flask, or the like.

[0076] As now can be appreciated, a conjugate of a magnetic particle and a
surface modifier having a specific affinity to selected target cells can
conveniently be used in processing of cells when the particle and
modifier are linked through a cleavable peptide bond specific to a
selected protease. While specific exemplary conjugates are described for
illustration purposes herein, in different applications variations and
modifications of the specifically disclosed examples may be possible, as
can be understood by those skilled in the art. For example, different
magnetic particles or different surface modifiers may be used in the
conjugates. The linker linking the modifier to the magnetic particle may
have a different structure and may include additional components, as long
as cleavage of the peptide bond will sever the link between the particle
and the modifier, and cleavage of the peptide bond can be catalyzed by
exposing the conjugate to the specific protease.

[0077] Conveniently, by selecting surface modifier that has higher
specific affinity to the target cells, cell processing efficiency and
effectiveness may be improved.

[0078] Also conveniently, a conjugate disclosed herein may be cleaved to
facilitate subsequent cellular processes, such as cellular adhesion.

[0079] In an exemplary embodiment, cell processing may be performed as
illustrated in the process S300 of FIG. 5. At S302, a conjugate is
attached to a target cell. The conjugate has a magnetic particle and a
surface modifier selected to have a specific binding affinity to the
target cell. The particle and modifier are linked through a cleavable
peptide bond. The target cell attached to the conjugate is then subject
to magnetic processing at S304. After magnetic processing, the peptide
bond is cleaved to separate the target cell from the magnetic particle at
S306. The target cell separated from the magnetic particle can then be
conveniently attached to a substrate at S308. The conjugate may be
conjugate 100. The peptide bond may be selected such that cleavage of the
peptide bond is catalyzed by a specific protease, such as thrombin. Thus,
severance of the link between the magnetic particle and the cell may be
effected by exposing the peptide bond to the specific protease. In this
embodiment, the surface modifier may be a glucosamine, glutamine,
galactose, or another small molecule that has specific affinity to a
given type of target cells. The magnetic particle may be a quantum dot or
a nanoparticle. For example, magnetite nanoparticles may be used. The
linker should be suitable for attachment to the magnetic particle, and
may include a terminal group that can bind with the surface of the
magnetic particle either by a chemical bond or by physical bonding. The
modifier and the linker may be chemically bonded, and may be provided in
a single molecule. The modifier and the linker may also be attached to
one another through physical bonding.

[0080] In another exemplary embodiment, a conjugate for attachment to a
cell is formed by linking a surface modifier to a magnetic particle
through a cleavable peptide bond. The surface modifier is selected to
have a specific affinity to the cell. The peptide bond is selected such
that cleavage of the peptide bond is catalyzed by a specific protease, so
that cleavage of the peptide bond can be conveniently effected by
exposing the conjugate to the specific protease. In this embodiment, the
protease may be thrombin. The surface modifier may be a glucosamine,
glutamine, galactose, or another small molecule that has specific
affinity to the cell. The magnetic particle may be a quantum dot or a
nanoparticle.

[0081] Suitable surface modifiers may be small molecules with a functional
group that has different binding affinities to surface receptors on
different types of cells. A larger difference in the binding affinities
to target cells and non-target cells may provide more selective
attachment to the cells, and thus increased processing efficiency.

[0082] The target cells may be any cells that have surface receptors for
specifically binding with the selected surface modifier. For example,
with a glucosamine as the surface modifier, insulin-secreting beta cells
may be the target cells as the glucosamine modifier has high binding
affinity to the Glut2 receptors on the cell surface. It has been found
that insulin-secreting beta cells attached with conjugates of magnetite
nanoparticle and glucosamine can be effectively separated from
surrounding (non-target) cells by applying a magnetic field to the cell
population. The cell population can thus be purified, for example, to
have up to 80% of insulin-secreting beta cells.

[0083] In at least some embodiments, when the exemplary conjugates are
used in cell processing, the linker in the conjugates, such as linker
106, should be selected so that the corresponding specific protease will
not adversely impact the viability of the cells when it is used to cleave
the linker, including not interfering with a subsequent attachment of the
cell to a substrate. Accordingly, the protease should be selected so that
it does not recognize the surface proteins on the cells, or at least the
important surface protein(s), such as a protein involved in the
subsequent substrate attachment process. In other words, the recognition
sequence for the protease should not be present on the surface of the
target cells and other useful cells in the cell mixture.

[0084] More generally, it should be understood that when used with cells,
the conjugates, particularly their surface materials and any portions of
the conjugates that may interact with the attached or surrounding cells,
should be formed with materials that are biocompatible with the cells and
will not have significant adverse effects such as toxic effects on the
cells.

[0085] The conjugates disclosed herein can find use in many different cell
processing applications. For instance, as discussed above, the conjugates
can be used in cell separation applications. As cell separation is a
common step in many biomedical and tissue engineering applications based
on cells, embodiments of the present invention are useful in such
biomedical applications.

[0086] Cell separation may be used to remove unwanted cells, which may
trigger the malfunction of the specified cells of interest. For example,
the presence of unwanted myoblasts or other cell types in a cardiomyocyte
population may hinder the synchronous beating behavior of cardiomyocytes.
In another example, unwanted kidney tubule epithelial cells would
transform into fibrotic cells when cultured along with fibroblasts.

[0087] Using the embodiments disclosed herein, insulin-secreting beta
cells may be conveniently separated, for example, from embryonic stem
cells (ESCs) such as after differentiation therefrom, from induced
pluripotent cells (iPS), or from adult stem cells such as bone marrow
mesenchymal stem cells (MSCs).

[0088] The conjugates disclosed herein can also be used in applications
utilizing a chromatography technique, such as a column chromatography
technique. An exemplary column chromatography technique is the expanded
bed absorption (EBA) technique.

[0089] Other applications and uses of the conjugates are also possible as
can be understood by those skilled in the art.

[0090] Exemplary embodiments of the present invention are further
illustrated with the following examples, which are not intended to be
limiting.

[0092] The iron oleate complex was dissolved in 1-octadecene (25 g), and
oleic acid (1.41 g, 5 mmol) or sodium oleate (1.52 g, 5 mM) was next
added. The mixture was heated to 320° C. and maintained at that
temperature for 1 h. The resulting black solution was cooled to room
temperature and 2-propanol was next added to precipitate the magnetic
particles. The particles were further centrifuged and washed with hexane
and ethanol, and redispersed in hexane or toluene. The resulting iron
oxide nanoparticles were used as the sample iron oxide nanoparticles in
other examples described herein, and are referred to as Sample I.

Example II

Synthesis of Peptide-Glucosamine

IIA. Conjugation of Maleimide to Glucosamine

[0093] The basic reaction for this synthesis procedure was as shown in
FIG. 2. A flame dried 5-mL reaction vial was charged with an aqueous
stock solution of glucosamine hydrochloride (5 mg in 0.25 mL, 0.021 mmol)
and a dry dimethylformamide (DMF, 0.25 mL) stock solution of
6-maleimidohexanoic acid N-hydroxysuccinimide ester (7 mg, 0.022 mmol)
under argon atmosphere, and cooled in an ice bath at 0° C. Dry DMF
(1 mL) was added dropwise, and the pH of the reaction was adjusted to 8
by carbonate buffer. The reaction mixture was stirred at 0° C. for
2 h under argon, and then brought to room temperature and stirred for
another 24 h under argon. DMF was removed under reduced pressure, and the
residue was dried under high vacuum to obtain a white residue, which was
referred to as Reagent 1 and was used directly in step IIB without
further purification.

IIB. Conjugation of Peptide to Maleimidoglucosamine

[0094] The basic reaction for the conjugation process was as shown in FIG.
3. A peptide (Reagent 2 as shown in FIG. 3) (17 mg, 0.02 mmol) was
dissolved in phosphate buffer (2 mL, pH 7.2), and was treated with
maleimidoglucosamine (Reagent 1). Reagent 2 includes the protease
recognition sequence of cys-Leu-Val-Pro-Arg-Gly-Ser-gly-cys-gly. The
reaction mixture was covered with an aluminum foil, and stirred under
argon for 24 h. The solution was purified by reverse phase recycling
high-performance liquid chromatography (HPLC) using a refractive index
(RI) detector, and freeze dried to obtain a white powder (20 mg, 82%)
product, referred to as Reagent 3 as shown in FIG. 3.

Example III

Conjugation of Glucosamine-Peptide Complex to Iron Oxide Particles

[0095] 15 mg of
O,O'-bis[2-(N-succinimidyl-succinylamino)ethyl]polyethylene glycol
(biNHS-PEG), a homobifunctional amine reactive crosslinker, was dissolved
in 100 μL of dimethylsulfoxide. This was added to the sample iron
oxide particles as produced in Example I. The mixture was sonicated for
30 min. Excess PEG linker was added to ensure that there were unreacted
NHS groups on the particle surface available for glucosamine conjugation
in the next step. The activated nanoparticles were then passed through a
PD-10 desalting column rinsed with 10 mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer. The
particles were collected and split into 2 separate vials.

[0097] Vial 2: 1.5 μmol of glucosamine was dissolved in 1 ml of 10 mM
HEPES buffer. This was mixed with the activated iron oxide particles
immediately and stirred overnight at 4° C.

[0098] The conjugated nanoparticles were centrifuged, and washed with 10
mM of HEPES using a microcentrifuge filter (molecular weight cutoff
(MWCO)=30 kDa)). The sample particles collected were used in the
following Examples.

[0099] Sample particles produced from Vial 1 are referred to as Sample
IIIA and sample particles produced from Vial 2 are referred to as Sample
IIIB herein.

Example IV

Synthesis of Glucosamine-Coated Iron Oxide Nanoparticles (Comparison)

[0100] Glucosamine was conjugated to sample iron oxide particles in two
steps. The synthesis route is illustrated in FIG. 6. First, sample iron
oxide nanoparticles were made hydrophobic via tetramethylammonium
hydroxide (TMAH). Next, glucosamine was coated on the surface of the
sample particles. Briefly, 1 mg of iron oxide nanoparticles were
precipitated and centrifuged by adding an equal volume ratio of ethanol.
0.5 mL of 1 M TMAH in H2O was then added to the black precipitate,
and the mixture was sonicated for 5-10 min. The mixture was left to stand
for another 10 min, and then 0.5 mL of acetone was added to precipitate
the particles. The particles were then redispersed in deionized water,
and washed with acetone.

[0101] To coat with glucosamine, sample nanoparticles dispersed in water
(1 mg in 250 μL) were added to 1 mg of glucosamine in 2 mL of
H2O. The solution remained clear, and was mixed overnight. The
solution was next centrifuged at 25000 g for 30 min, and the particles
were collected and redispersed in water. This was repeated once, followed
by redispersion in water. The resulting particles remained stable in
deionized water for weeks, and will be referred to as Sample IV herein.

[0103] 1 ml of crosslinked glutathione-capped ZnS-CdS-CdSe
(cGSH-ZnS-CdS-CdSe) quantum dot (referred to as "QD595") solution (1
mg/ml) was diluted to 20 ml with 100 mM borate buffer (pH 8.0). 10 mg of
N-hydroxysuccinimide (NHS) and 20 mg of
1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) were
freshly dissolved in 2 ml of 100 mM borate buffer, and were immediately
added to the QD595 solution with stirring. 1 ml of D-glucosamine
dissolved in 100 mM borate buffer to a concentration of 1 mg/ml was
added. After incubation overnight, the system was quenched with a 50 mM
glycine buffer (pH 7.5). Glucosamine-conjugated QDs were purified by
ultrafiltration with a membrane of 50 KDa molecular weight cutoff (MWCO).

[0104] The resulting glucosamine-conjugated QDs will be referred to as
Sample V.

Example VI

Attachment of Sample Conjugates to Cells

[0105] Sample V conjugates were mixed with insulin-secreting beta cells to
attach the conjugates to the cells, by rocking the mixture in a rocker at
a speed of 30 rpm/min at 37° C. (5% CO2).

[0106] Fluorescence micrographs of the test samples showed a strong
presence of the glucosamine-QDs595 (λem=595 nm) on the
surface of insulin-secreting beta cells. Representative confocal
microscopic images of the tested samples are shown in FIGS. 10 and 11.

[0107] For comparison, QDs595 without glucosamine were also mixed
with insulin-secreting beta cells. It was observed that uptake of the QDs
without glucosamine by the cells was non-specific. A representative
confocal microscopic images of the tested sample is shown in FIG. 12.

[0108] Flow cytometry results indicated that 38% of the cells were labeled
as "QD-positive" when Sample V was used FIG. 13 shows the QD uptake
distribution for Sample V in a mixture of fibroblasts and
insulin-secreting beta cells incubated with Sample V, as analyzed by flow
cytometry showing auto-fluorescence.

[0109] In comparison, QD update was substantially negative when QDs
without glucosamine was used, as can be seen in FIG. 14 which was for the
control mixture of fibroblasts and insulin-secreting beta cells incubated
with bare QDs.

[0110] The positive and negative fractions from the flow cytometry for
Sample V were further analyzed for specific genes using real-time
polymerase chain reaction (RT-PCR) with gene-specific primers. The
fibroblast used contained neomycin gene incorporated in its genome.
Hence, the specific markers for these fibroblasts were neomycin and CD90.
In comparison, the specific gene targets for insulin-secreting beta cells
were insulin and Glut2.

[0111] The sample cells were subject to ribonucleic acid (RNA) isolation
and two-step RT-PCR as follows. The total RNA was isolated from the cells
using the Genelute RNA isolation kit (Sigma®, USA) according to the
manufacturer's protocol. 3 μg of DNase I (Rnase free, Invitrogen®)
treated total RNA was reverse transcribed into complementary
deoxyribonucleic acid (cDNA) with Superscript III (Invitrogen, USA) for
90 min at 42° C. PCR was performed with Advantage 2 Taq polymerase
(BD biosciences®, USA). Gene-specific primers were designed from the
available sequences from the Singapore National Center for Biotechnology
Information gene databank. RT-PCR was conducted in Bio-Rad iCycler®
using TaqMan assay for the specific genes obtained from Applied
Biosystems®, USA.

[0112] Real-time PCR results indicated that the "QD-negative" fraction and
"QD-positive" fraction had strong expressions of the markers associated
with fibroblasts and insulin-secreting beta cells, respectively. FIG. 15
shows the representative PCR results, where the gene expression in the
initial mixture was used for normalization (i.e. 1-fold).

[0113] Separate tests for cell attachments were also performed with Sample
IIIA, Sample IIIB, and Sample IV as the respective conjugates.

[0114] Test results showed that conjugates of glucosamine and iron oxide
nanoparticles exhibited high binding efficiency to insulin cells, and
provided up to 80 to 85% of insulin cells recovery in a magnetic column
based cell separation process.

[0115] Glucosamine's affinity to Glut2 receptors was tested by eluting
glucosamine-bound fibroblasts and insulin cells with different
concentrations of glucose. The elution profiles of fibroblasts and
insulin-secreting beta cells were different, as shown in FIGS. 16 and 17.
FIG. 16 shows the results of real-time PCR analysis of the cells
separated using Sample IV conjugates. The gene expressions in the initial
cell input was used for normalization (i.e. 1-fold). The flow-through
(negative) fraction and the bound (positive) fraction were analyzed for
the insulin-secreting beta cell specific gene expression using gene
specific primers. FIG. 17 shows the cumulative elution profiles of
fibroblasts and insulin cells incubated with Sample IV conjugates under
different glucose concentration. It indicated that the binding affinities
of fibroblasts and insulin cells to Sample IV conjugates were different.
Fibroblasts could be eluted at a lower concentration of glucose (10 mM),
while insulin-secreting beta cells required a higher concentration of
glucose (20 mM). This result indicated that insulin-secreting beta cells
had a higher affinity to glucosamine, as compared to fibroblasts. Glut2
was expressed on insulin-secreting beta cells but not on fibroblasts,
which bonded to glucosamine through Glut1. It can thus be expected that
Glut2 has a high affinity to glucosamine.

[0117] The cells were dispersed to separate individual cells by adding
trypsin. The separated cells were washed with phosphate buffered saline
(PBS) (twice) and incubated with Sample IV conjugates produced in Example
IV for 1 h in the binding buffer, which was formed of 2% of bovine serum
albumin (BSA) and 1 mM ethylenediaminetetraacetic acid (EDTA) in PBS. The
cells were passed through a magnetic column attached to a magnet. The
column was washed with washing buffer (PBS containing 2% of BSA). The
flow-through solution was collected as the negative binding fraction,
while the bound fraction was collected upon removal of the magnetic
force.

[0118] In separate tests, cells labeled with Sample V conjugates
(QD595) or cytotracker were suspended in PBS containing 5% FBS. The
artificially mixed populations of insulin cells (50%) and fibroblasts
(50%) were used to test cell separation in a flow cytometry platform with
Sample V conjugates.

[0119] Samples collected at different stages of cell separations were
analyzed using a 3-laser LSR II FACS® analyzer from BD Biosciences,
USA.

[0120] Separate tests for cell separation were also performed with Sample
IIIA as the attached conjugates.

[0121] Using fluorescently labeled fibroblasts and unlabeled
insulin-secreting beta cells in cell separation tests, the selective
attachment properties of the glucosamine conjugates were verified by flow
cytometry. Upon binding of the cells to the magnetic column, the cells
were washed with 10 mM glucose (to first remove most of the weakly bound
fibroblasts), followed by the elution of the remaining cells bound to the
column.

[0122]FIG. 18 shows the profiles of the cytometry analysis of the sample
mixture of cells prior to separation. The mixture of cells contained
mouse fibroblasts labeled with red fluorescence artificially mixed with
insulin-secreting beta cells. Insulin-secreting beta cells were separated
using Sample IV conjugates. FIG. 19 shows the results of cytometry
analysis of the flow-through fraction of the cells that passed the
magnetic column after cell separation, which, as can be seen, contained
mostly fibroblasts (˜85%). FIG. 20 shows the results of cytometry
analysis of the bound fraction of cells after cell separation, which
contained mostly insulin-secreting beta cells (˜75%). The flow
cytometry results indicated that 85% of the fibroblasts were recovered in
the 10 mM glucose wash fraction, and that the bound fraction contained
mainly (˜75%) unlabeled cells (insulin-secreting beta cells).

[0123] Tests were also conducted to enrich insulin-secreting beta cells
from whole pancreas of pigs. Pancreatic islets contained mainly 3 types
of cells, alpha cells (˜15% of islet cells, identified by glucagon
expression), insulin-secreting beta cells (˜80% of islet cells,
identified by insulin), and Glut2 and delta cells (˜3% of islet
cells, identified by somatostatin expression). Islets were isolated from
the pig pancreas and treated with collagenase to form single cells. These
cells were incubated with Sample IV conjugates. The conjugate-bonded cell
fraction ("enriched") was analyzed for gene expression by real-time PCR.
The results are shown in FIG. 21. The enriched fraction was found to have
strong expressions of the markers associated with beta cells. The
real-time PCR results showed that the enriched population contained
mainly the insulin- and Glut2-expressing insulin-secreting beta cells.
Furthermore, the absence of expression of somatostatin and glucagon
confirmed that the enriched insulin-secreting beta cell population was
not contaminated by the surrounding islet cells, such as alpha cells and
delta cells.

Example VIII

Cleavage of Links Between Cells and Magnetic Particles

[0124] Tests were conducted to confirm that the links between iron oxide
nanoparticles and the cells could be cleaved by exposure to thrombin. In
these tests, sample cells bonded to iron oxide particles by way of Sample
IIIA conjugates were incubated with 50 units of thrombin (total
volume=0.5 ml) at 37° C. for 30 min. The suspension was then
exposed to magnetic field and the unbonded fraction was collected.

Example VIII

Attachment of Cells to Substrate

[0125] Insulin cells were incubated with Sample IIIA and IIIB conjugates
respectively. Sample IIIA conjugates contained a thrombin-specific
peptide linking glucosamine to the iron oxide particle. Sample IIIB
conjugates did not contain a peptide linker. The cells attached with the
conjugates were subject to magnetic field separation and collected. As
shown in FIG. 22, the percentage of cells attached with the conjugates
was similar for both Samples IIIA and IIIB.

[0127] The collected cells bonded to Sample IIIA were incubated with 50
units of thrombin for 30 min at 37° C. as described in Example
VII, and then subject to further magnetic field separation. The
flow-through fraction that contained the released cells was collected,
and cultured on tissue culture plates (substrate).

[0128] Representative images of the respective culture plates taken after
24 h of culturing are shown in FIGS. 23 (for Sample IIIB) and 24 (for
Sample IIIA), respectively. It was observed that the separated insulin
cells attached to Sample IIIB failed to adhere and proliferate, like the
control cells that were not subject to the cell separation procedure. It
was also observed that the cells released from the magnetic particles in
Sample IIIA conjugates successfully adhered to the culture substrate.

[0129] As used herein, and unless otherwise specifically indicated to the
contrary, the term "comprise", including any variation thereof, is
intended to be open-ended and means "include, but not limited to."

[0130] When a list of items is given herein with an "or" before the last
item, any of the listed items or any suitable combination of the listed
items may be selected and used. For any list of possible elements or
features provided in this specification, any sublist falling within a
given list is also intended. Similarly, for any range provided, any
subrange falling within a given range is also intended.

[0131] Of course, the above described embodiments are intended to be
illustrative only and in no way limiting. The described embodiments are
susceptible to many modifications of form, arrangement of parts, details
and order of operation. The invention, rather, is intended to encompass
all such modification within its scope, as defined by the claims.